The present invention relates an oligomer array and a method of producing an oligomer array, such as for instance a peptide array. In addition also the use of such an oligomer arrays for detecting a binding partner is described.
Oligomer arrays are well-known within the prior art and refer to the entirety of the oligomers that are combinatorially synthetized onto a carrier or a substrate, respectively (consisting of individual molecular components, such as e.g. monomers). Herein they bind covalently on a carrier being configured as spots. The synthesis of these oligomers in general resides on the chemical principles of the solid-phase synthesis. The realization of the parallel solid-phase synthesis, in particular the transport of the monomers to the respective synthesis location, herein is of decisive importance.
Such oligomers immobilized on an oligomer array comprise all kinds of oligomers that can be synthetized combinatorially from several components. Examples of such components comprise amino acid derivatives and nucleotide derivatives, such as deoxyribonucleotide derivatives and ribonucleotide derivatives.
The oligomer arrays mentioned above are of interest in particular with respect to the detection/determination of binding partners. Herein for instance a suitable binding partner is determined by means of hybridization. The existence of a respective binding event for instance may establish the inhibition of the effect of a biomolecule, whereby the utilization of the respective inhibiting oligomer as a medicament is made possible. The parallel search of as many oligomers as possible being potentially biological active therefore is of high importance within medicine and adjacent branches. To make possible a parallel searching of a large number of oligomers, in particular the preparation of ultra high dense oligomer arrays is of interest within which more than 106 oligomers per square centimeter or spots/cm2, respectively are present on the carrier immobilized.
Within the prior art there is known a variety of different methods for producing oligomer arrays. These methods for preparation of polynucleotides in particular are characterized by a simplified production, when compared to the peptide synthesis, this being the case since substantially only four components are utilized. For instance the synthesis of a library of oligonucleotide sequences can be initially performed by means of an oligonucleotide synthesizer that is highly parallel including an intermediate PCR increase. The finally synthesized strands subsequently are coupled into microparticles (beads) so that per microparticle there is a kind of an oligonucleotide sequence. Thereafter the microparticles are arranged on a surface within array format, and the location of the microparticles and thee sequences of the respective oligonucleotides are determined using a special hybridization technique. However, this method is limited to oligonucleotides (Gunderson K. L. et al.; Genome Research; 14(5) (2004), pp. 870-877).
Further methods for generating oligonucleotides comprise phage display and ribosomal display methods. Herein synthetically generated oligonucleotides are fused with the gene of a phage shell protein so that after transfection of a bacterium each bacterium “packs” a different kind of phage that only differs within the sequence of the peptides fused with the phage shell protein (cf. Smith, G. P. filamentous fusion phage—novel expression vectors that display cloned antigens on the virion surface; Science 228 (1985) pp. 1315-1317).
The combinatorial peptide synthesis using semiconductor chips for instance may be a high voltage CMOS chip the surface of which is divided into different electrodes (M. Beyer et al., Science, 318 (5858), 1888, 2007). By programming the chip individual electrodes can be activated selectively. By means of the electric fields generated thereby loaded particles that serve as carriers for the monomers are deposited at the electrodes thereof at precise locations. The synthesis of the oligomer arrays may either be performed directly on the chip surface or on a target carrier (for instance a glass object slider). To this end particle patterns generated on the CMOS chip are transferred onto the target carrier by means of an electric field.
In addition lithographic methods are known by means of which spot densities of up to 106 spots/cm2 can be obtained (Fodor S. P. A. et al.; light-directed, spatially addressable parallel chemical synthesis. Science 251, pp. 767-773 (1991); and Legutki J.-B. Nat. Commun. 5, 4785, 2014). Using light masks protective groups are separated according to the generated light pattern that allows the binding of a subsequent amino acid parts. Apart from the necessity of a precise positioning of the synthesis carriers such a lithographic method has a further disadvantage that for each monomer there must be performed a separate coupling reaction. This inevitably leads to secondary reactions that prevent that peptide arrays of sufficiently good quality are commercially available (Palloys J. P. et al.; individually addressable parallel peptide synthesis on microchips; Nature Biotechnology 20 (2002), pp. 922-926).
In the case of the combinatorial laser fusing (CLF) monomer-containing particles are fixed directly on a synthesis carrier by means of laser irradiation. Herein a laser beam is guided across the carrier to selectively melt the particles (Maerkle F. et al., High-Density Peptide Arrays with Combinatorial Laser Fusing, Advanced Materials, Volume 26 (2014), pp. 3730-3734).
A further method relates to a xerographic method wherein the toner particles of a 24 color laser printer are printed that each contain an amino acid component for the combinatorial synthesis. Such a method is for instance described in WO 00/35940.
The lithographic method described above needs a variety of coupling cycles, if an oligomer array and, in particular a peptide array, shall be generated thereby. The number of coupling cycles can be computed from N×Y, wherein N refers to the number of the different monomers and Y refers to the length of the oligomers. If for instance an array of 15 mer peptides shall be synthesized within which 20 amino acids are used, than the number of coupling cycles is computed to be 300. Due to this reason lithographic methods up to now only are used successfully for the synthesis oligonucleotide arrays. Therefore currently there is no other commercially available method by means of which spot densities of more than 40,000 spots/cm2 can be realized with sufficiently high quality. A further technical problem are the complicated and costly devices that are necessary for the lithographic and also for all other methods.